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Issue 99, 2014
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Ligase-assisted, upconversion luminescence resonance energy transfer-based method for specific and sensitive detection of V600E mutation in the BRAF gene

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Abstract

The most common BRAF mutation, V600E, accounts for a variety of cancers. Here we report a highly specific and sensitive method for the detection of the V600E mutation. The detection scheme is based on luminescence resonance energy transfer (LRET) between upconversion nanoparticles (UCNPs) and an intercalating dye, SYBR Green I. Target DNA serves as the template for two DNA probes, one of them covalently attached to UCNPs, to be ligated into a hairpin-forming DNA strand, which brings SYBR Green I close to the upconversion nanoparticles. The number of the resulting DNA strand is amplified through thermal cycling. The degree of LRET is correlated to the amount of the initial DNA targets. Factors affecting the detection specificity and sensitivity, including ligation temperature, amount of ligase, and number of thermal cycles, have been investigated to optimize the performance of the detection method. The method can easily differentiate the V600E mutation from the wild-type sequence with a mutant-to-wild-type ratio of 1 : 1000. A detection limit of 1 femtomole BRAF V600E mutation is achieved.

Graphical abstract: Ligase-assisted, upconversion luminescence resonance energy transfer-based method for specific and sensitive detection of V600E mutation in the BRAF gene

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Article information


Submitted
10 Sep 2014
Accepted
21 Oct 2014
First published
23 Oct 2014

RSC Adv., 2014,4, 56235-56240
Article type
Paper
Author version available

Ligase-assisted, upconversion luminescence resonance energy transfer-based method for specific and sensitive detection of V600E mutation in the BRAF gene

P. Wang and P. Zhang, RSC Adv., 2014, 4, 56235
DOI: 10.1039/C4RA10181B

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